Airway epithelial cells are located at the interface between the environment and underlying lung tissue. The lung epithelium forms a physical barrier against environmental insults and participates in defense against invading bacteria, viruses and fungi. Although the contribution of epithelial cells to antibacterial and antiviral immunity is well established, the role of lung epithelium in immunity to fungi is poorly understood. This proposal fills this knowledge gap by studying how the airway epithelium orchestrates host defense against medically important fungi. To test the hypothesis that airway epithelial cells underpin the generation of adaptive immunity to fungi, 3 aims are proposed. First, we will assess fungal clearance from the lung where airway epithelial cell function is selectively ablated (Aim 1) during murine infection with Blastomyces dermatitidis, Histoplasma capsulatum, or Aspergillus fumigatus. Second, by using one of the fungal infection models from Aim 1, we will identify the epithelial cell receptors that recognize fungi and trigger production of inflammatory mediators (Aim 2). Toll-like (TLR) and C-type lectin (CLR) receptors mediate fungal recognition and members of these receptor families are expressed on epithelial cells. To dissect the mechanisms of fungal recognition, we will co-culture primary airway epithelial cells from TLR- and CLR-deficient mice with B. dermatitidis, H. capsulatum or A. fumigatus and measure chemokines in the supernatant. This approach will unveil defects in chemokine production that stem from TLR and/or CLR deficiencies. Third, we will define how epithelial cells impact the development of downstream immune responses, such as the priming of CD4+ T helper (Th) cells (Aim 3) that is paramount in antifungal immunity. To address this question, we will analyze the phenotype and function of TCR transgenic, fungus-specific Th cells in infected mice genetically engineered to exhibit impaired airway epithelial cell function. Epithelial cells may influence Th cell responses indirectly by controlling the properties of dendritic cells (DC) that initiate Th cell responses. Thus, we will analyze the numbers, phenotype and function of lung DC ex-vivo upon infection of epithelial cell-impaired mice to ascertain how epithelial cell-derived chemokines affect the recruitment and activation of DC. This work will take advantage of cutting-edge tools including transgenic mice, cell sorting, microfluidics and live cell imaging and will be performed in a leading laboratory and center for the study of lung immunity to fungi. In addition to investigating vital research questions, the work will provide the applicant state-of the-art research training and didactic teaching skills. Upon completion of the training program, we will know how airway epithelial cells sense fungal pathogen(s), how the resulting soluble signals influence DC that shape the development of CD4+ T cell responses, and how the sum of these events promote the resolution of infection. Understanding how respiratory epithelial cells mobilize antifungal immunity will have implications for the development of antifungal vaccines and immune-based therapies in immune-compromised patients, where fungal infections are common and often fatal.